This invention relates to methods and apparatus for assaying biological samples to which a reagent is added and particularly, to computer-controlled methods and apparatus for such assaying.
Assaying processes are well known in which a reagent is added to a sample, and measurements of the sample and reagent are made to identify sample attributes stimulated by the reagent. For example, one such assay process concerns determining in a chromogenic assay the amount of an enzyme present in a biological sample or solution. Such assays are based on the development of a colored product in the reaction solution. The reaction develops as the enzyme catalyzes the conversion of a colorless chromogenic substrate to a colored product.
In such assays it is often required to determine the enzymatic activity of a number of samples and at one or more dilutions. Enzymatic reactions characteristically proceed at a constant rate provided substrate is present in a large molar excess, i.e., the concentration of substrate does not limit the rate of reaction. With such kinetic parameters, it may be convenient to set up several reaction solutions separately in the wells of a microtiter plate, for example, carrying out each reaction for a predetermined constant amount of time and stopping the reactions while they are still in a linear range of the assay. With each of the so-called end-point reactions stopped, no further color development occurs and the reaction solutions in the separate wells of the microtiter may be read at any convenient time.
Plate readers which automatically read the intensity of a colored solution in an array of wells are known. Also, plate readers which measure the amount of fluorescence in a well of a microtiter plate are known.
Classically, assays of the above-described type are performed by a laboratory worker who prepares the sample, manually adds a precise amount of reagent to the sample, and then measures the result at one or more preselected times after the reagent addition. This classical approach is very time consuming for the laboratory worker and additionally, when the stimulated reaction yields time-varying results, precise timing on the part of the laboratory worker is required. If such timing is not properly performed, erroneous assay results may occur.
One known laboratory device for partially automating tests to detect fluorescence as a measurable attribute is the Fluoroskan II. The Fluoroskan II includes a plate carrier system to hold a sample-containing plate having a plurality, e.g., 96, of sample-containing wells. A laboratory worker places a portion of the sample into some or all of the wells of the plate, and then adds reagent to the sample-containing wells. The plate is then placed in the Fluoroskan II which automatically measures the fluorescence of the samples in the wells. Although this known apparatus has proven valuable for fluorescence testing, some problems, which were also inherent in the classical testing, still remain. For example, the addition of reagent by the laboratory worker still requires a large amount of laboratory work time. Also, since the measured results of reactions may be dependent on the time elapsed since reagent addition, the first samples to receive reagent may have progressed past the point of meaningful reaction results by the time all samples have received reagent. Further, some reactions complete so quickly that it is nearly impossible to add reagent to a sample, move the sample plate to the assay apparatus and move the sample to a measurement position before the reaction has run to completion.
A need exists to rapidly screen compounds to determine their effect on a protein's function such as cell surface proteins like ion channels and receptors, the regulation of which surface proteins can be important in treating certain disease states. Such cell surface proteins permit intracellular transduction of extracellular signals. These cell surface proteins, by transmitting information regarding extracellular stimuli via specific intracellular pathways, induce an appropriate cellular response to external stimuli. The response may be immediate and transient, slow and sustained, or some mixture thereof. By virtue of an array of varied membrane surface proteins, normal (i.e., non-diseased) cells and tissue are exquisitely sensitive to their environment. Compounds which are capable of potentiating or inhibiting activation of voltage-dependent calcium channels are believed to be useful in treating a variety of diseases including certain cardiovascular and nervous system disorders. Similarly, compounds which can affect the functioning of cell surface receptors may be beneficial in the treatment of certain other diseases. Thus, it is desirable to identify compounds capable of activating, potentiating or inhibiting such cell surface proteins using functional assays. Such assays require study of the kinetics of the reaction in real time due to their transient nature. Heretofore, such assays have required a substantial amount of time from highly skilled researchers and technicians to conduct and record the results of individual assays. Also, with respect to functional assays for cell surface proteins which utilize electrophysiological or fluorescence imaging techniques, laboratory equipment which is required is often extremely costly.
A need also exists for an automated analysis apparatus which is capable of automatically adding reagent to contained samples and measuring reaction results to minimize lab worker time requirements, and to provide accurate measurement of time varying reactions. It would also be very advantageous, inasmuch as drug companies now typically screen thousands to tens of thousands of compounds in the hope of finding a desired activity, to be able to provide a rapid automated method for assaying a compound or a series of compounds for ability to affect cell surface proteins, such as ion channels and receptors or cytoplasmic receptors, where a plurality of tests may be conducted seriatim and the results recorded with no, or only occasional, intervention by the researcher.